Organ transplantation has become the treatment of choice for various diseases associated with organ failure (Platt J L (1998) Nature 392:11-17; Auchincloss H Jr, and Sachs D H (1998) Ann Rev Immunol 16:433-70). However, the supply of organs from organ donors falls far short of meeting the rapidly increasing demand (Hammer C, Linke R, Wagner F, and Diefenbeck M (1998) Int Arch Allergy Immunol 116:5-21). In the United States, only 5% of the patients on various waiting lists for organ transplants ever receive the appropriate organs. One attractive approach to overcoming this shortage is to use animals as a source of organs for transplantation (Greenstein J L and Sachs D H (1997) Nat Biotechnol 15:235-238). The transplantation of organs or cells between members of different species is called xenotransplantation. Non-human primates are the closest biological relatives of human beings; therefore, their organs are most similar to humans, anatomically, physiologically and biochemically. In fact, organs from chimpanzees and baboons have been shown in clinical studies to exhibit extended xenograft survival following rather simple immunosuppression procedures (Bailey L L, Nehlsen-Cannarella S L, Concepcion W, and Jolley W B (1985) JAMA 254(23):3321-3329; Starzl T, Marchioro T L, and Peters G (1964) Transplantation 2:752-776). However, the large-scale production of these endangered non-human primates for the purpose of securing organs for transplantation is considered unethical and socially unacceptable (Cortesini R (1998) Transplant Proc 30(5):2463-2464). Furthermore, the transmission of pathogens from primates to humans is well documented, and pathogen-free primates are extremely difficult to raise (Allan J S (1995) Nat Medicine 2(1):18-21). In addition, a significant obstacle to the widespread adoption of xenotransplantation is the immunological incompatibility between nonprimate animals and humans, which results in strong host rejection responses to the xenotransplanted organ. Overcoming these host rejection responses is essential to enable widespread use of nonprimate animal organs in xenotransplantation.
The first immune barrier to xenograft survival is hyperacute rejection, which may occur within minutes after revascularization of an organ (Rosenberg J C, Hawkins E, and Rector F (1971) Transplantation 11(2):151-157). The rejection is induced by the activation of the host (recipient) complement cascade upon the binding of recipient xenoreactive natural antibodies to the xenograft. The major xenograft antigen (or "xenoantigen") responsible for the hyperacute rejection response has been identified as a carbohydrate epitope, Gal.alpha.1,3Gal.beta.1,4GlcNAc (referred to herein as "the .alpha.Gal epitope"). The .alpha.Gal epitope forms glycoconjugates on the cell surface of animal organs and cells (Thall A, and Galili U (1990) Biochemistry 29: 3959-3965; Good A H, Cooper D K C, Malcolm A J, Ippolito R M, Koren E, Neething F A, Ye Y, Zuhdi N and Lamontagne L R (1992) Transplantation Proceedings 24:559-562). The .alpha.-Gal epitope is universally present in the animal kingdom, with the exception of humans and Old World monkeys who lack the galactosyltransferase responsible for the epitope synthesis. Conversely, in normal human serum there are significant amounts of naturally occurring anti-.alpha.Gal antibodies, which constitute approximately 1-3% of total IgG molecules and 3-5% of total IgM (Rother R P and Squinto S P (1996) Cell 86:185-188). Recent studies suggest that anti-.alpha.Gal IgM, rather than IgG, is responsible for the hyperacute rejection response observed in organ xenotransplantation (Kroshus T J, Bolman R M III, and Dalmasso A P (1996) Transplantation 62:5-12).
The second immunological barrier to xenografts is termed delayed or vascular rejection. Although a detailed mechanism has yet to be elucidated, vascular endothelium cells are considered to be the target for immune activation through antibody-dependent cytotoxicity mediated by NK cells and macrophages (Lawson J H, and Platt J L (1996) Transplantation 63:303-310). In contrast to hyperacute rejection, both IgG and IgM induce vascular rejection effectively, and complement is apparently not involved. These antibodies may represent xenoreactive natural antibodies whose specificities have not yet been characterized. Finally, the cellular immune response constitutes the last barrier for xenotransplantation. The mechanism involved is probably similar to that observed in allograft rejection, but with more potent responses.
Although immune responses to xenografts are divided into three stages, most research and clinical strategies thus far developed have been aimed only at the first stage, hyperacute rejection. One approach to reducing hyperacute rejection involved circulating human recipient blood over an .alpha.Gal immunoadsorbent column to remove anti-.alpha.Gal antibodies prior to transplantation of the xenograft (Taniguchi S, Neethling F A, Korchagina E Y, Bovin N, Ye Y, Kobayashi T, Niekrasz M, Li S, Koren E, Oriol R, Cooper D K C (1996) Transplantation 62:1379-1384). However, while the procedure was successful in reducing the concentration of anti-.alpha.Gal antibodies in the human blood, the reduction in antibody levels was transient, and was often followed by a rapid rebound within days. A more attractive alternative is to alter the .alpha.Gal epitope on the donor organ by expressing or knocking out specific glycosyltransferase/glycosidase activities in organ donor animals (Osman N, McKenzie I F C, Ostenried K, Ioanou Y A, Desnick R J, and Sandrin M S (1997) Proc Natl Acad Sci 94:14677-14682; Koike C, Kannag, R, Takuma Y, Akutsu F, Hayashi S, Hiraiwa N, Kadomatsu K, Muramatsu T, Yamakawa H, Nagai T, Kobayashi S, Okada H, Nakashima I, Uchida K, Yokoyama I, and Takagi H (1996) Xenotransplantation 3:81-86) (see FIG. 1). A third approach is to generate transgenic animals that express human complement regulatory proteins, such as DAF, CD46 and CD59 (Zaidi A, Schmoeckel M, Bhatti F, Waterworth P, Tolan M, Cozzi E, Chavez G, Langford G, Thiru S, Wallwork J, White D, and Friend P (1998) Transplantation 65: 1584-1590). In this approach, although binding of xenoreactive antibodies still takes place, the presence of these regulatory proteins may prevent complement-induced cell lysis. Data from several studies seem to suggest that a combination of different approaches may be required to efficiently inhibit hyperacute rejection associated with xenograft transplantation.
In recent years, a consensus has emerged that the domestic pig may represent a good alternative to nonhuman primates as a donor of organs for transplantation. Porcine and human organs have similar sizes and cardiac output efficiencies. Pigs are relatively easy and inexpensive to raise in large numbers. Furthermore, pigs can be more easily raised in sterilized environments than nonhuman primates, and the use of pigs as organ donors produces fewer ethical concerns. The greater phylogenetic distance between pigs and humans means it is less likely that xenografts of pig organs or cells would impose any realistic risk of transmitting an infectious organism of epidemiological significance to the human population. However, the immunological and physiological incompatibility between pigs and humans remains a significant obstacle to the widespread use of pig organs for transplantation.
Advances in the field of xenotransplantation have brought to light the intriguing prospect of using porcine blood for use in xenotransfusions. Serologically, human blood and pig blood have a number of important features in common, including a similar hematocrit, blood volume, and number of blood groups. Biochemically, human hemoglobin has been expressed in pig and functions normally in vivo (Rao M J, Schneider K, Chait B T, Chao T L, Keller H, Anderson S, Manjula B N, Kumar R, and Acharya A S (1994) Artif Cells Blood Substit Immobil Biotechnol 22:695-700). However, surface antigens on porcine red blood cells ("pRBCs") are significantly different from those identified on human red blood cells, and are recognized by antibodies in human serum. Thus, pRBCs would certainly be short lived if injected into human blood circulation. Since donor endothelium is not involved, the reactions in human immunological responses to pRBCs are probably different from the reactions associated with organ transplantation. One may gain some insight into the human immune responses to pRBCs from a consideration of the fate of mismatched red cells upon allo-transfusion in humans. There are two principal mechanisms of in vivo red cell destruction in allo-transfusion (Transfusion Reactions in Applied Blood Group Serology, Chapter 36, 4.sup.th Edition (1998) (Eds. Issitt, P D and Anstee D J) Montgomery Scientific Publications, Durham, N.C.). The first mechanism, termed intravascular destruction, is triggered by antibody binding and complement activation. Red cells are thus rapidly lysed and release hemoglobin into the blood stream. IgG molecules may play a major role in intravascular destruction, since many IgM antibodies against blood groups are cold-reactive and do not bind efficiently to red cells in vivo. The second mechanism for clearing red cells in vivo is extravascular destruction, in which intact red cells are removed from circulation primarily by macrophages in the liver and spleen. In this case red cells are coated with IgG, but not at a high enough concentration to activate the complete complement cascade. Under these conditions, the complement cascade is usually halted at the C3 stage.
As is the case with other porcine cells thus far studied, pRBCs carry the .alpha.Gal epitope on the cell surface, and this epitope accounts for the major reaction observed with xenoreactive human natural antibodies. However, there are data suggesting that porcine red cells may also carry additional xenoantigens other than the .alpha.Gal epitope (MacLaren L, Lee T D G, Anderson D, Nass M, and McAlister V C (1998) Transplant Proc 30:2468). The natures of these non-.alpha.Gal xenoantigens and corresponding human xenoreactive antibodies have not been explored. Identification of these non-.alpha.Gal epitopes and finding ways to reduce or eliminate host immune rejection responses directed to these epitopes is important to fully realize the potential benefits of using porcine organs in xenotransplantation.